Application of a Ultra Performance Liquid Chromatography Method in the Determination of DNA Quality and Stability

Stabilization of Plasmid DNA and Lipid-Based Therapeutics as Dehydrated Formulations

“…Furthermore, we have observed that trehalose-containing formulations maintain DNA integrity for 9 weeks at higher temperatures (e.g., 40, 50, and 60 °C) in the presence of DTPA, and also resulted in better preservation of the initial biological activity (Figs. 2b, 3a, b, [110]). In our hands, however, pure sucrose-containing formulations did not protect naked plasmid DNA in terms of biological activity and DNA integrity even at 40 °C (Figs.…”

“…An effective strategy is to incorporate chelating agents into DNA preparations [82]. Indeed, our recent investigation has shown the great effectiveness of the ion-chelating agent DTPA to counteract the damaging effect of ROS (e.g., lost in SC) in lyophilized DNA-based formulations during acute lyophilization [115] and during storage [110,178]. DTPA-prevented ROS levels increase in the presence of Fe 2+ (400 μM), which is consistent with the fact that its affinity for iron (log K a of Fe 2+ = 16, [189]) is larger than the affinity of iron for double-stranded DNA (log K a of Fe 2+ = 4.73, [190]).…”

“…Unfortunately, the undesirable complications associated with gel staining (e.g., ethidium bromide) such as background variability, nonlinearity in staining, and the requirement of corrections for more accurate determination of SC content due to the distinct intercalating affinities of ethidium bromide for the different species [176] have encouraged the development of more accurate chromatographic methods: ultra performed liquid chromatography (UPLC; [110]) and anion-exchange high-pressure liquid chromatography (AE-HPLC; [173]). For instance, UPLC has proven to be a valuable tool to monitor the distribution of plasmid isoforms in dried samples subjected to extreme conditions such as metal contamination and storage under accelerated conditions [110]. Certainly, maintenance of plasmid DNA integrity (i.e., ≥ 80 % SC as recommended by FDA, [160]) is an important structural characteristic that will have an impact not only on bulk production but also long-term stability (e.g., in the dried state), efficacy, and eventually clinical approval.…”

“…Furthermore, it is important to mention that plasmid DNA homogeneity (i.e., most plasmid is in its SC form) is a major concern to pharmaceutical manufacturers since efficacy and stability depend, in part, on the topology of DNA (i.e., comparative topology in the relative amounts of SC; open circular, OC; and linear forms) [110,140,[172][173][174]. It is generally recognized that via single-strand break, the SC form (used as a standard measure of DNA quality [160]) can convert to OC form, and further degradation can produce undesirable amounts of the linear form that has been associated with lower levels of expression once transfected [174,175].…”

“…Despite the growing body of research on nonviral vector dehydration, to date, lyophilization studies have mostly focused on the stability of vectors (e.g., naked DNA [91,[110][111][112][113][114], lipoplexes [83,86,[115][116][117], polyplexes [85,88,99,109,118,119], and lipopolyplexes [95,120]) during acute freeze-drying stress. Considering the complexity involved in the stabilization of individual vectors (e.g., naked DNA, lipid) as dehydrated formulations, this chapter addresses these challenges separately.…”